Clarithromycin-Loaded Submicron-Sized Carriers: Pharmacokinetics and Pharmacodynamic Evaluation

The current study aims to improve clarithromycin bioavailability and effectiveness in complicated intra-abdominal infection management. Therefore, clarithromycin-loaded submicron dual lipid carriers (CLA-DLCs) were developed via hot high shear homogenization technique and evaluated for colloidal parameters, release behavior, stability study, and in-vitro antibiofilm activity. Bioavailability and therapeutic efficacy of optimized formulation on hampering cytokines storm induction was determined in E. coli-induced peritonitis. The developed CLA-DLCs (particle size 326.19 ± 24.14 nm, zeta potential −31.34 ± 2.81 mV, and entrapment efficiency 85.78 ± 4.01%) exhibited smooth spherical shapes and sustained in vitro release profiles. Long-term stability study of optimized CLA-DLCs ensured maintenance of colloidal parameters for 1 year at room temperature. In vitro antimicrobial studies revealed 3.43-fold higher anti-biofilm activity of CLA-DLCs compared with clarithromycin. In addition, the relative bioavailability of CLA-DLCs was enhanced 5.89-fold compared to pure drug in rats. The remarkable decrease in microbial burden in blood as well as tissues, along with oxidative stress markers (lipid peroxidation, myeloperoxidase activity, and carbonylated protein level) and immunological markers (total leukocyte count, neutrophil migration, NO, TNF-, and IL-6) on treatment with CLA-DLCs enhanced the survival in a rat model of peritonitis compared with the pure drug and untreated groups. In conclusion, CLA-DLCs hold promising potential in management of intra-abdominal infections and prevention of associated complications.


Introduction
Complicated intra-abdominal infections (cIAIs) are a complex fatal clinical challenge. The most alarming causative biofilm-forming pathogens, such as Staphylococcus aureus, Haemophilus influenzae, Streptococcus pneumoniae, and Enterobacteriaceae are of particular concern, as they are accountable for flare-ups of infections [1]. Thus, polymicrobial etiology and difficulty in diagnoses present an additional obstacle in cIAI management [2]. Furthermore, development of multidrug resistance makes cIAI management formidable and poses a significant burden on quality of life and the health-care system [3]. The associated complications, such as uncontrolled immune responses, leading to systemic inflammatory condition due to severe infection can lead to organ dysfunction, sepsis, or septic shock. Thus, peritonitis and coexisting alarming inflammatory responses are the vital cause of mortality in immunocompromised and critically ill patients [4]. Therefore, an urgent need exists to develop alternative antimicrobial therapy for management of cIAIs.
Several studies recommend macrolides alone or in combined antibiotic therapy as the treatment of choice due to their broad spectrum of antimicrobial activity [5,6]. Among macrolides, clarithromycin (CLA) is a drug of choice due to higher stability in acidic milieu Lyophilized CLA-DLCs were analyzed for particle size, polydispersity index (PDI), and zeta potential utilizing dynamic laser light scattering spectroscopy (Nano ZS, Malvern, UK). The quantitative values of measurement parameters were as follows: measurement temperature of 25 • C, a medium viscosity of 0.8872, mPa s, and a medium refractive index of 1.330. Aqueous dispersion of lyophilized CLA-DLCs in distilled water was prepared by ultrasonicating samples for 30 s before evaluation.

Entrapment Efficiency
Supernatants collected after separation of CLA-DLCs pellet (as explained in preparation section) were estimated at 207 nm for CLA content using UV/Visible spectrophotometer. Entrapment efficiency was estimated by using the following equation [33]: Surface morphology of optimized CLA-DLCs was analyzed utilizing scanning electron microscope (MIRA3 TESCAN). CLA-DLCs were mounted on aluminum stubs using double adhesive tape and sputter-coated with gold palladium alloy under an inert atmosphere of argon before capturing images at suitable magnification.
Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of CLA, stearic acid, compritol, physical mixture of components, and optimized lyophilized formulation were acquired utilizing Bruker EQUINOX 55 FTIR spectrophotometer. Samples were inflexibly clenched in contrast to ATR lens and examined in transmission mode over wavelength range from 4000 to 600 cm −1 .
Thermograms of CLA, stearic acid, compritol, physical mixture of formulation excipients, and optimized lyophilized formulation were recorded to determine drug-lipid compatibility by differential scanning calorimeter (DSC-204 F1 phoenix, Republic of Korea). Samples were placedin standard aluminum pans and heated at a pace of 10 • C min −1 over a range of 30-300 • C under continuous purging of nitrogen at 60 mL min −1 .
X-ray diffraction patterns of CLA, stearic acid, compritol, physical mixture of drug and excipients, and optimized lyophilized CLA-DLCs were recorded by employing powder X-ray diffractometer (XPERT-PRO, PAN analytical, The Netherlands) with CuKα radiation (45 Kv,40 mA) in the diffraction range of 0-80 • , 2θ.

Drug Release Studies
The assessment of CLA release from CLA-DLCs was determined in pH progressive media (HCl buffer pH 1.2 for 2 h, followed by phosphate buffer pH 6.8) utilizing dialysis bag diffusion method. CLA-DLCs equivalent to 10 mg of CLA dispersed in 1.5 mL distilled water was filled in dialysis bag (12)(13)(14) and immersed in dissolution medium (200 mL) kept at 37 ± 2 • C and 100 rpm. Sink condition was maintained by adding 0.1% w/v sodium lauryl sulfate. Sampling (3 mL) was executed at 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 h, with consecutive replacement with equal volume of fresh buffer (37 • C). Samples were centrifuged, and supernatants collected were analyzed for CLA content. Drug release kinetics was determined by plotting obtained data according to different release kinetics models. The best fit model was determined on the basis of the numerical value of the calculated regression coefficient.

Stability Studies
Optimized lyophilized CLA-DLCs stored in glass vials with polystyrene screw caps were examined for stability according to guidelines issued by ICH for Zones III and IV. Samples were kept at different storage conditions, including room temperature (25 ± 2 • C and 60 ± 5% relative humidity (RH)) and cool conditions (4 ± 2 • C and 65 ± 5% RH) for 12 months. Samples were evaluated for colloidal stability (particle size, PDI, and zeta potential) and entrapment efficiency at intervals of 0, 1.5, 3, 6, and 12 months.

In Vitro Antimicrobial Activity Minimum Inhibitory Concentration (MIC) Determination
The minimum amount of CLA and CLA-DLCs needed to inhibit growth of E. coli (MTCC code 433) was determined by broth dilution method. Concisely, sterile nutrient broth was inoculated with bacterial suspension (100 µL, equivalent to 0.5 McFarland standard) in culture tubes with subsequent addition of different concentration of test samples equivalent to 0.25, 0.50, 0.75, 1, 2, 4, and 6 µg/mL of CLA, respectively. The turbidity, an indication of bacterial growth in culture tubes, was analyzed at 600 nm after an incubation period of 24 h at 37 • C.

Minimum Biofilm Inhibitory Concentration (MBIC) Evaluation
Briefly, broth inoculated with E. coli (adjusted to 0.5 Mc Farland standards) for 24 h at 37 • C was decanted to remove non-adherent bacteria. Biofilms adhered to walls and bottoms of culture tubes were washed twice with sterile distilled water to draw out free bacteria. Subsequently, biofilm from the surface of tubes was detached, collected, and treated with various concentrations of CLA or CLA-DLCs equivalent to 25,30,35,40,70,100, and 120 µg/mL of clarithromycin. Treated biofilms were withdrawn and incubated in fresh broth for 24 h at 37 • C. The presence of turbidity measured at 600 nm indicated presence of viable organisms [9].

In Vivo Studies
Wistar rats (200 ± 20 g) acquired from the institutional animal house were kept on standard animal diet at 22 ± 2 • C and 55 ± 5% RH on a 12 h dark/light cycle. In vivo

Pharmacokinetic Evaluation
Overnight-fasted Wistar rats were randomized into two groups (n = 6) for single dose oral bioavailability study. Animals of Groups I and II received CLA suspension (5 mg/kg) and CLA-DLCs (equivalent to 5 mg/kg of CLA), respectively. Blood samples (250 µL) were withdrawn from rat tail vein into heparinized tubes at 0.5, 1, 2, 4, 6, 8, 12, and 24 h postdosing. Blood loss was compensated by injecting equivalent volume of normal sterile saline solution. Later, blood was centrifuged at 5000 rpm for 10 min at 4 • C to segregate plasma and analyzed for drug content by HPLC method [9]. Pharmacokinetic parameters were estimated using WinNonlin software (5.1:Pharsight, Mountain View, CA, USA).
Microbial count in blood and tissue homogenates was determined by plating samples on agar plates, followed by incubation at 37 • C for 48 h and counting the colonies formed.

Histopathological Evaluation
Isolated organs (lungs and spleen) were cut into small pieces of 0.5 cm 3 and stored in 10% neutral buffered formalin. Samples were processed by serially dehydrating in increasing concentrations of ethanol, followed by clearing in xylene and embedded in paraffin using Leica EG 1160 Embedding machine. Transverse sections of 3 µm were cut, deparaffinized, hydrated to distilled water, and processed for hematoxyline-eosin staining. Tissue sections were observed under microscope, and images were captured at 40× magnification.

Statistical Analysis
Data were presented as mean ± SD (n = 3). Statistical analysis using one-way ANOVA followed by Bonferroni's multiple comparison test was performed utilizing GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). p < 0.05 was considered as statistically significant in all studies.

Formulation Optimization
The remarkable variation in particle size and encapsulation efficiency was noticed on varying amount of lipids due to the differences in their HLB values. The average particle size and entrapment efficiency upon varying the compritol 888 ATO:stearic acid ratio from 1:2 to 2:1 ranged from 315.34 ± 24.15 nm to 579.24 ± 28.87 nm and from 61.64 ± 6.47% to 85.78 ± 4.01% (Table 2), respectively. The complex nature of compritol and its less perfect orientation leaves more space for drug loading in comparison with stearic acid. In addition, the long chain length of behenic acid in compritol 888 ATO enhances the intermolecular entrapment of the drug by interchain intercalation and intrusion in crystal lattice of stearic acid [36]. The enhanced CLA entrapment may be attributed to the fact that an increase in compritol 888 ATO content facilitates the reduction of the melting point of stearic acid and leads to massive disturbances in the lattice crystal order in stearic acid which bestows more space for drug loading in submicron lipid carriers [23,37] (Table 2). In addition, the relative increment of compritol content might have increased the viscosity of organic phase leading to faster solidification of submicron particles, which hindered diffusion of the CLA to the external phase [38]. However, an additional increment in compritol content of more than 50% w/w of total lipid had no improvement in CLA loading, manifesting saturation of the compritol effect [39]. Further increase in compritol content led to increase in particle size and PDI values, respectively along with decrease in entrapment efficiency ( Table 2). Table 2. Effect of diverse variables on quality parameters, i.e., particle size, polydispersibility index, zeta potential, and entrapment efficiency.
On increasing the pluronic F-68 concentration from 0.1% to 0.5% w/v, an increase in encapsulation efficiency and a decrease in particle size were noticed, respectively (Table 2). However, further increase in pluronic F-68 concentration (1% w/v) reduced size and entrapment efficiency ( Table 2). The results established the need for notable amounts of surfactant to form stable colloidal systems and impede its solubilizing ability at higher concentrations [9,35,40]. The polydispersity index (PDI) of all batches prepared on varying surfactant concentrations and types of surfactants also followed a similar trend as particle size.
On varying the drug amount from 10 mg to 20 mg, an increase in encapsulation efficiency was observed. However, a further increase in drug amount (30 mg) led to a decrease in encapsulation efficiency due to a higher diffusion of the drug into the continuous phase as a relatively smaller proportion of lipid was present in comparison with drug amount.
An increment in external phase volume (50 mL) contributed to a decrease in encapsulation efficiency, with increase in particle size and PDI, respectively (Table 2). Insufficient amplitude of shear encountered at higher volume (50 mL) might have contributed to the decrease in encapsulation efficiency and rise of particle size and PDI, respectively [9,27].
An increment of sonication time from 7 min to 10 min had significantly lowered particle size and PDI along with improvement in encapsulation efficiency. However, further increment in duration of sonication (15 min) imparted higher shear stress, which might have ruptured the surfactant barrier and made the system thermodynamically unstable (Table 2) [27,41].
Zeta potential, a quantitative measurement of surface charge governs the physical stability of the system. Batches prepared on altering various formulation and process variables have zeta potentials in the range of −17.81 ± 2.19 mV to −31.34 ± 2.87 mV. Zeta potential of approximately ±30 mV generates columbic forces of higher magnitude, which overcome the van der Waals forces of attraction and represent higher physical stability of colloidal system [41].
Thus, on the basis of the outcomes, the CLA-DLC formulation (F2) produced utilizing compritol 888 ATO and stearic acid in a 1:1 ratio, drug amount of 20 mg, pluronic F-68 (0.5% w/v) as surfactant, 25 mL external phase volume, and sonication for 10 min was utilized for further studies.

Solid State Characterization
SEM microphotographs of CLA-DLCs demonstrated the formation of spherical-shaped submicron particles with smooth surfaces and uniform size distribution ( Figure 1A).  Figure 1B).DSC thermogram of clarithromycin showed a sharp characteristic peak at 220 °C, while stearic acid and compritol exhibited broad peaks at 63.58 °C and 73 °C, respectively. Optimized formulation showed a broad endothermic peak at 80° C. The non-existence of an endothermic peak of clarithromycin in the DSC thermogram confirmed the homogeneous dispersion of clarithromycin in optimized formulation ( Figure 1C).
X-ray diffractogram of pure clarithromycin showed sharp and intense peaks at 9.40°, 11.40°, 12.32°, 13.67°, 15.11°, 17.22°, 18.98°, and 20.40° (2θ). Sharp diffraction peaks confirmed the crystalline nature of the drug [9]. The diffractogram of stearic acid exhibited broad halo peaks at diffraction angles of 21.36° and 25.81°. Similarly, the diffractogram of compritol showed broad halo peaks at diffraction angles of 21.16° and 29.95°. The physical mixture of clarithromycin with compritol and stearic acid exhibited distinctive peaks  Figure 1B). DSC thermogram of clarithromycin showed a sharp characteristic peak at 220 • C, while stearic acid and compritol exhibited broad peaks at 63.58 • C and 73 • C, respectively. Optimized formulation showed a broad endothermic peak at 80 • C. The non-existence of an endothermic peak of clarithromycin in the DSC thermogram confirmed the homogeneous dispersion of clarithromycin in optimized formulation ( Figure 1C).
X-ray diffractogram of pure clarithromycin showed sharp and intense peaks at 9.  Figure 1D). Figure 2 represents the biphasic drug release pattern, i.e., an initial burst release (~16%, lasting 2 h in simulated gastric pH) followed by gradual drug release over 24 h (~90%) under bio-relevant conditions of the gastro-intestinal tract. Initial rapid release might have contributed to prompt diffusion and desorption of surface-adsorbed or weakly bound drug or partial film defects emerged during lyophilization [35]. The steady decline in the drug release rate might be ascribed to intrinsic drug solubility, path length of drug-depleted layers across which the drug travelled, rigidity of lipoidal barrier, and/or diffusion from dialysis membrane [27]. The results indicated that formulation would exhibit prolonged drug release behavior in simulated intestinal conditions. Materials 2023, 16, x FOR PEER REVIEW 9 of 18 of clarithromycin in the diffractogram. However, the CLA-DLC diffractogram showed no existence of any distinctive diffraction patterns of clarithromycin in the optimized formulation indicating a uniform molecular dispersion of drug and loss of crystallinity of drug in LCs ( Figure 1D). Figure 2 represents the biphasic drug release pattern, i.e., an initial burst release (~16%, lasting 2 h in simulated gastric pH) followed by gradual drug release over 24 h (~90%) under bio-relevant conditions of the gastro-intestinal tract. Initial rapid release might have contributed to prompt diffusion and desorption of surface-adsorbed or weakly bound drug or partial film defects emerged during lyophilization [35]. The steady decline in the drug release rate might be ascribed to intrinsic drug solubility, path length of drug-depleted layers across which the drug travelled, rigidity of lipoidal barrier, and/or diffusion from dialysis membrane [27]. The results indicated that formulation would exhibit prolonged drug release behavior in simulated intestinal conditions.

Stability Studies
The F2 formulation stored at cool conditions and room temperature manifested no considerable alteration in particle size, PDI, zeta potential, or entrapment efficiency after 12 months of storage (p < 0.05) (Figure 3). This indicated that formulation variables were suitably optimized to retain pharmaceutical properties of CLA-DLCs for effective long-term use. Thus, results of stability study recommend storage of CLA-DLCs at room temperature conditions [27]. The physical appearance of CLA-DLCs did not show any noticeable changes at different environmental conditions.

Stability Studies
The F2 formulation stored at cool conditions and room temperature manifested no considerable alteration in particle size, PDI, zeta potential, or entrapment efficiency after 12 months of storage (p < 0.05) (Figure 3). This indicated that formulation variables were suitably optimized to retain pharmaceutical properties of CLA-DLCs for effective long-term use. Thus, results of stability study recommend storage of CLA-DLCs at room temperature conditions [27]. The physical appearance of CLA-DLCs did not show any noticeable changes at different environmental conditions.

In Vitro Anti-Bacterial Studies
Lower MIC value of CLA-DLCs (0.75 μg/ ml) revealed higher effectiveness of formulation in inhibiting E. coli growth compared with free drug (6 μg/ ml). Similarly, 3.43-fold lower MBIC of CLA-DLCs has assured its higher efficacy in inhibiting E. coli biofilm formation compared with the pure drug ( Figure 4). The enhanced bactericidal activity of CLA-DLCs might have contributed to their ability to deliver their pay load into host cells through different mechanisms, e.g., contact release, adsorption, and endocytosis. The physiological resemblance of CLA-DLCs to membrane lipids might have improved uptake mechanism by fusion with cell wall or cell membrane. The enhanced cellular uptake and subsequent extended release of encapsulated CLA might have effectively improved the antibacterial effect of CLA-DLCs [9,[43][44][45].

In Vitro Anti-Bacterial Studies
Lower MIC value of CLA-DLCs (0.75 µg/mL) revealed higher effectiveness of formulation in inhibiting E. coli growth compared with free drug (6 µg/mL). Similarly, 3.43-fold lower MBIC of CLA-DLCs has assured its higher efficacy in inhibiting E. coli biofilm formation compared with the pure drug ( Figure 4). The enhanced bactericidal activity of CLA-DLCs might have contributed to their ability to deliver their pay load into host cells through different mechanisms, e.g., contact release, adsorption, and endocytosis. The physiological resemblance of CLA-DLCs to membrane lipids might have improved uptake mechanism by fusion with cell wall or cell membrane. The enhanced cellular uptake and subsequent extended release of encapsulated CLA might have effectively improved the antibacterial effect of CLA-DLCs [9,[43][44][45].  3.6. Pharmacokinetic Profile Figure 5 represents plasma concentration versus time profiles for the single oral dose of CLA-DLCs formulation and CLA suspension, while corresponding pharmacokinetic parameters are presented in Table 3. CLA-DLCs attained 2.32-fold higher Cmax compared with CLA suspension. Submicron size as well as hydrophobic surface of CLA-DLCs might have aided their enhanced uptake and improved plasma drug con-  Figure 5 represents plasma concentration versus time profiles for the single oral dose of CLA-DLCs formulation and CLA suspension, while corresponding pharmacokinetic parameters are presented in Table 3. CLA-DLCs attained 2.32-fold higher Cmax compared with CLA suspension. Submicron size as well as hydrophobic surface of CLA-DLCs might have aided their enhanced uptake and improved plasma drug concentration. Delayed Tmax, i.e., 4 h, also confirmed in vivo extended drug release profile of CLA-DLCs similar to in vitro release profile. CLA plasma concentration declined after 2 h, indicating rapid systemic clearance, which was further ascertained by lower MRT (Table 3). The elevated t 1/2 (2.87-fold) and MRT (1.84-fold) of CLA-DLCs further confirmed the extended absorption and release of drug from CLA-DLCs in systemic circulation. The remarkable improvement in AUC 0-24h (p < 0.05) with CLA-DLCs (~5.93-fold) compared with CLA dispersion might be credited to improved systemic absorption of CLA-DLCs through paracellular and transcellular pathways via enterocytes, M-cells, and gut-associated lymphoid tissues or transport via triglyceride rich lipoproteins [33,35,46].  Figure 5 represents plasma concentration versus time profiles for the single oral dose of CLA-DLCs formulation and CLA suspension, while corresponding pharmacokinetic parameters are presented in Table 3. CLA-DLCs attained 2.32-fold higher Cmax compared with CLA suspension. Submicron size as well as hydrophobic surface of CLA-DLCs might have aided their enhanced uptake and improved plasma drug concentration. Delayed Tmax, i.e., 4 h, also confirmed in vivo extended drug release profile of CLA-DLCs similar to in vitro release profile. CLA plasma concentration declined after 2 h, indicating rapid systemic clearance, which was further ascertained by lower MRT ( Table 3). The elevated t1/2 (2.87-fold) and MRT (1.84-fold) of CLA-DLCs further confirmed the extended absorption and release of drug from CLA-DLCs in systemic circulation. The remarkable improvement in AUC0-24h (p < 0.05) with CLA-DLCs (~5.93-fold) compared with CLA dispersion might be credited to improved systemic absorption of CLA-DLCs through paracellular and transcellular pathways via enterocytes, M-cells, and gut-associated lymphoid tissues or transport via triglyceride rich lipoproteins [33,35,46].   Values are expressed as mean ± SD, n = 3.

Pharmacodynamic Study Effect of CLA-DLCs on Oxidative Stress Biomarkers
The quantitative results of bacterial cultures of blood and tissue homogenate samples of CLA-DLCs treated group showed remarkable reduction in microbial load (p < 0.05) compared with the pure drug and saline-treated groups (Table 4). An approximately 10 4 -fold lower microbial load in the blood, lung, and liver whereas 10 3 -fold change in spleen of CLA-DLC treated animals confirmed higher antimicrobial potential of CLA-DLCs compared with the pure drug. The endotoxin administration manifested elevation of oxidative stress markers in lung, liver, and spleen ( Figure 6). Pure drug and CLA-DLCs were found remarkably efficacious in bringing down the LPO, MPO, and carbonylated protein level in all tissues compared with the control (p < 0.05). The results revealed CLA-DLCs were more effective in lowering oxidative stress markers and bacterial count compared with the pure drug (p < 0.05) due to facilitated distribution of formulation in reticuloendothelial system rich organs, such as the lungs, liver, and spleen, major reservoirs of bacterial load, as is evident from pharmacokinetic studies [27,47] (Figure 6). Significant elevation of serum total leukocyte count, neutrophils, lymphocytes, TNF-α, IL-6, and NO level in the control group on intraperitoneal administration of LPS was observed. Contrarily, treatment groups (CLA suspension and optimized formulation) showed a noticeable reduction (p < 0.05) (Figure 7). CLA ability to reduce circulating Significant elevation of serum total leukocyte count, neutrophils, lymphocytes, TNF-α, IL-6, and NO level in the control group on intraperitoneal administration of LPS was observed. Contrarily, treatment groups (CLA suspension and optimized formulation) showed a noticeable reduction (p < 0.05) (Figure 7). CLA ability to reduce circulating LPS by attenuating as well as killing bacteria might have contributed to a reduction in the level of inflammatory markers (TNF-α and IL-6) in plasma and infiltration of inflammatory markers (neutrophils and lymphocytes) in tissues [47,48]. CLA-DLCs treatment facilitated significant reduction in mean values of total leukocyte count, neutrophil, lymphocytes, TNF-α, IL-6, and NO level (p < 0.05) compared with drug and control (Figure 7). The unique characteristics of CLA-DLCs, i.e., submicron size, ease of adherence or diffusion to bacterial cell, and sustained drug release significantly inhibited bacterial growth and impeded the bacterial endotoxin-induced systemic inflammatory conditions. In addition, CLA-DLCs delivered higher CLA plasma concentrations upto 24 h and better distribution of CLA to tissues compared with the pure drug. Thus, CLA-DLCs significantly hampered the cascade of events induced by bacterial infection during peritonitis management.

Histology
Signs of lung injury-interstitial edema, alveolar thickening, and small peribronchial cellular aggregates with a large number of lymphocytes in interstitium and alveoliwere observed in lung tissues of the control group ( Figure 8A). Histology of spleen

Histology
Signs of lung injury-interstitial edema, alveolar thickening, and small peribronchial cellular aggregates with a large number of lymphocytes in interstitium and alveoliwere observed in lung tissues of the control group ( Figure 8A). Histology of spleen tissues also demonstrated excessive infiltration of neutrophils, contributing to the disruption of normal architecture of the spleen ( Figure 8D). The results of histology are in correlation with the level of oxidative markers and microbial count in respective tissues [49]. The endotoxins released and presence of E. coli in different tissues might have triggered the infiltration of neutrophils with elevation of MPO and LPO level, prominent mediators of tissue inflammation [48,49].  On the contrary, the CLA-treated group showed lower infiltration of neutrophils in tissues in comparison with the control (Figure 8B,E). The CLA-DLC-treated group showed significantly reduced neutrophil infiltration in the spleen and lung tissues compared with the drug and control groups ( Figure 8C,F). Improved distribution and residence time of drug via CLA-DLCs might have provided the protection to tissues from damage by reducing microbial load, oxidative stress, and neutrophil migration [9,27,35].

Conclusions
CLA-DLCs were successfully fabricated using a hot homogenization method with higher entrapment efficiency and prolonged drug release behavior. Enhanced mean residence time and half-life of drug had remarkably enhanced bioavailability as well as On the contrary, the CLA-treated group showed lower infiltration of neutrophils in tissues in comparison with the control (Figure 8B,E). The CLA-DLC-treated group showed significantly reduced neutrophil infiltration in the spleen and lung tissues compared with the drug and control groups ( Figure 8C,F). Improved distribution and residence time of drug via CLA-DLCs might have provided the protection to tissues from damage by reducing microbial load, oxidative stress, and neutrophil migration [9,27,35].

Conclusions
CLA-DLCs were successfully fabricated using a hot homogenization method with higher entrapment efficiency and prolonged drug release behavior. Enhanced mean residence time and half-life of drug had remarkably enhanced bioavailability as well as tissue distribution. Formulation specifically reduced bacterial burden in organs, such as the lungs, liver, and spleen, where abscesses were present. CLA-DLCs have been found experimentally to be an ideal delivery system for mitigating the severity of infection by mopping up the endotoxins, preventing growth of E. coli, and reducing oxidative stress and tissue damage. Concisely, CLA-DLCs hold great promise as an effective therapy with the possibility of reduced dosing frequency for management of peritonitis and avoidance of further complications, such as septic shock.
Author Contributions: Conceptualization, methodology, investigation, supervision, writing-reviewing and editing, M.S.; investigation, data curation, and writing, R.R.; reviewing and editing, R.S.C.; reviewing and editing, V.S. All authors have read and agreed to the published version of the manuscript.
Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional Animal Ethics Committee of Banasthali Vidyapith (protocol code BU/3418/16-17 and date of approval was 8 October 2016).

Informed Consent Statement:
Not applicable for studies as not involving humans.
Data Availability Statement: Submitted manuscript contains complete research data. No data has been shared in any repository.